This invention relates to a reproducible method for visualizing proteins which have been separated by gel electrophoresis. More particularly, it relates to a method which involves the steps of fixing, staining and exposure to a metal complexing agent of an electrophoresed gel in order to produce a highly sensitive negative stain.
The employment of one and two dimensional gel electrophoretic separations of proteins have made possible the analysis of complex biological processes. A prevailing and predominant problem associated with the aforesaid analysis is the absence of a rapid, self limiting, sensitive, and reproducible method for visualizing the separated proteins.
Various methods referenced hereinafter suffer from one or more deficiencies in these parameters.
1. Fazekas de St. Groth, S.; Webster, R. G.; and Datyner, A. (1963) . New staining procedures for quantitative estimation of proteins on electrophoretic strips. Biochem. Biophys. Acta 71:377-391--an early paper which describes the use of Coomassie Brilliant Blue R250 and Procion Brilliant Blue RS in staining proteins separated by cellulose acetate strip electrophoresis.
2. Meyer, T. S. and Lamberts, B. L. (1965) Use of Coomassie Brilliant Blue R250 for the electrophoresis of microgram quantities of parotid saliva proteins on acrylamide gel strips. Biochem. Biophys. Acta 107:144-145--an early paper which describes the use of Coomasie Brilliant Blue R250 to stain acrylamide gels.
3. Davis, B. J. (1964) Disc electrophoresis-II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427--a paper commonly referred to for Amido Schwartz (Napthol Blue Black, C.I. 20470) staining of proteins in acrylamide gels.
4. Gorovsky, M. A.; Carlson, K. and Rosenbaum, J. L. (1970) Simple method for quantitative densitometry of polyacrylamide gels using Fast Green. Anal. Biochem. 35:359-370--an early paper which describes the use of Fast Green to stain proteins in acrylamide gels. The staining responses of proteins is the most linear with respect to protein concentration.
5. Merril, C. R.; Switzer, R. C.; and Van Keuren, M. L. (1979) Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain. Proc. Natl. Acad. Sci. USA 76:4334-4339--an early paper which describes highly sensitive silver stain for proteins on acrylamide gels; currently the most sensitive method to detect `cold` protein.
6. Dzandu, J. K.; Deh, M. E.; Barratt, D. L.; and Wise, G. E. (1984) Detection of erythrocyte membrane proteins, sialoglycoproteins, and lipids in the same polyacrylamide gel using a double-stain technique. Proc. Natl. Acad. Sci. USA 81:1733-1737--this paper describes a double stain technique to visualize sialoglycoproteins and lipid in polyacrylamide gels.
7. Fairbanks, G.; Steck, T. L.; and Wallach, D. F. H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606-2617--an early paper which describes the use of periodic acid-Schiff reagent (PAS) to stain glycoproteins in acrylamide gels.
Currently, sensitive work is accomplished with the aid of radiolabeled aminoacids. Since radiolabeled amino acids cannot be used for all types of sensitive work, e.g., sensitive work involving human tissue or proteins lacking the particular amino acid, it is highly desirable to have a staining technique which provides a highly sensitive negative stain. Until now, the most sensitive staining method to date was generally considered to be the silver stain. This method, however, entails substantial amounts of time and considerable care to perform.
The staining procedure herein disclosed and claimed relies on the use of planar dyes in combination with a complexing metal salt to differentiate between the matrix and the protein containing spot.